Nodule Worm Infection in Humans and Wild in : Cryptic Species in a Newly Identified Region of Human Transmission

Ria R. Ghai1, Colin A. Chapman2,3, Patrick A. Omeja3, T. Jonathan Davies1, Tony L. Goldberg3,4* 1 Department of Biology, McGill University, Montreal, Quebec, Canada, 2 Department of Anthropology and McGill School of Environment, Montreal, Quebec, Canada, and Wildlife Conservation Society, Bronx, New York, New York, United States of America, 3 Makerere University Biological Field Station, Fort Portal, Uganda, 4 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

Abstract

Introduction: Soil-transmitted helminths (STHs) are a major health concern in tropical and sub-tropical countries. Oesophagostomum infection is considered endemic to West but has also been identified in Uganda, East Africa, among primates (including humans). However, the taxonomy and ecology of Oesophagostomum in Uganda have not been studied, except for in chimpanzees (Pan troglodytes), which are infected by both O. bifurcum and O. stephanostomum.

Methods and Findings: We studied Oesophagostomum in Uganda in a community of non-human primates that live in close proximity to humans. Prevalence estimates based on microscopy were lower than those based on polymerase chain reaction (PCR), indicating greater sensitivity of PCR. Prevalence varied among host species, with humans and red colobus (Procolobus rufomitratus) infected at lowest prevalence (25% and 41% by PCR, respectively), and chimpanzees, olive (Papio anubis), and l’hoest monkeys (Cercopithecus lhoesti) infected at highest prevalence (100% by PCR in all three species). Phylogenetic regression showed that primates travelling further and in smaller groups are at greatest risk of infection. Molecular phylogenetic analyses revealed three cryptic clades of Oesophagostomum that were not distinguishable based on morphological characteristics of their eggs. Of these, the clade with the greatest host range had not previously been described genetically. This novel clade infects humans, as well as five other species of primates.

Conclusions: Multiple cryptic forms of Oesophagostomum circulate in the people and primates of western Uganda, and parasite clades differ in host range and cross-species transmission potential. Our results expand knowledge about human Oesophagostomum infection beyond the West African countries of and , where the parasite is a known public health concern. Oesophagostomum infection in humans may be common throughout Sub-Saharan Africa, and the transmission of this neglected STH among primates, including zoonotic transmission, may vary among host communities depending on their location and ecology.

Citation: Ghai RR, Chapman CA, Omeja PA, Davies TJ, Goldberg TL (2014) Nodule Worm Infection in Humans and Wild Primates in Uganda: Cryptic Species in a Newly Identified Region of Human Transmission. PLoS Negl Trop Dis 8(1): e2641. doi:10.1371/journal.pntd.0002641 Editor: Aaron R. Jex, University of Melbourne, Australia Received July 29, 2013; Accepted November 28, 2013; Published January 9, 2014 Copyright: ß 2014 Ghai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant: CAC, and Alexander Graham Bell Award: RRG) and the Fonds de Recherche du Quebec Nature et Technologies International Internship award, as well as by NIH grant TW009237 as part of the joint NIH- NSF Ecology of Infectious Disease program and the UK Economic and Social Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction a genus of nodule-causing worms with L3 larvae that are infective via ingestion after 4–7 days [4–7]. The human burden of Soil-transmitted helminths (STHs) are parasitic that Oeosphagostostomum infection is considered localized to West Africa, cause infection via eggs and larvae, which are shed in feces and specifically the countries of Togo and Ghana [5,8,9]. persist in the soils of tropical and sub-tropical countries [1]. STHs A variety of mammals, including , ruminants [10,11], and infect over one billion people worldwide [2] and may cause a non-human primates are frequently parasitized by Oesophagosto- combined disease burden as substantial as that caused by malaria mum. Infections in wild primates appear to be asymptomatic; or tuberculosis [3]. Nevertheless, these parasites are largely clinical signs and mortality due to Oesophagostomum have only been neglected in research, perhaps in part because the diseases they recorded in captive settings [10,12]. Eight species of Oesophagos- cause are suffered by the world’s most impoverished populations tomum have been recorded in wild primates, of which the three [1]. Although roundworms (Ascaris lumbricoides), hookworms (Necator most common, O. bifurcum, O. stephanostomum, and O. aculeatum, are americanus and Ancylostoma duodenale) and whipworms (Trichuris able to infect humans [5,7,11,13]. Of these, O. bifurcum appears to trichiura) are of global importance, other ‘‘lesser’’ parasites are be the only species to regularly parasitize humans, with human localized to specific regions [1]. This includes Oesophagostomum spp., infections by other species considered incidental [5,9]. In Togo

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Author Summary Methods Nodule worms infect the gastrointestinal tracts of a Ethics Statement number of mammalian species, including humans and Prior to data collection, all protocols were reviewed and other primates. This study sought to identify the species of approved by the Uganda National Council for Science and nodule worms causing infections within and around an Technology and the Uganda Wildlife Authority, as well as by the East African national park in Uganda where monkeys and Institutional Review Board and the Care and Use apes co-occur and overlap with people. Some primates, Committees of McGill University and the University of Wiscon- particularly those traversing large distances in small sin-Madison. Due to low literacy rates, oral informed consent was groups, were most susceptible to nodule worm infection. obtained from all adult subjects and a parent or guardian of all Additionally, molecular analyses identified three separate minor participants by trained local field assistants and documented groups of nodule worm that could not be distinguished by witnessed notation on IRB-approved enrollment forms. based on microscopic examination of their eggs. One of these groups was found in humans as well as other primates and had not previously been genetically charac- Study Site and Sample Collection terized. These results suggest that certain types of nodule Kibale National Park (0u139–0u419N, 30u199–30u329E) is a 2 worm may be restricted to particular hosts, while others 795 km semi-deciduous protected area in Western Uganda. may be transmitted among primates, including humans. research has occurred in Kibale for over four decades, Nodule worms are currently thought to be a human health focusing on chimpanzees and red colobus monkeys (Procolobus concern only in some West African countries. This research rufomitratus) [29,30]. As a result, a number of primate groups are suggests that nodule worms have a broader geographic habituated to human presence, and many individuals are impact in humans than previously appreciated. recognizable based on a combination of physical attributes and collars affixed as part of a larger project on primate health and conservation [31]. Samples from monkeys in the Kanyawara area of Kibale and Ghana, the majority of human Oesophagostomum cases occur National Park were collected from red-tailed guenons (Cercopithecus within endemic foci [5,8] and affect 20% and 90% of the ascanius), blue monkeys (Cercopithecus mitis), l’hoest monkeys population, respectively, with prevalence highest in rural areas (Cercopithecus lhoesti), grey-cheeked mangabeys (Lophocebus albigena), [12,14,15]. The only known species to cause infection within these olive baboons (Papio anubis), red colobus, and black and white countries is O. bifurcum, which also infects the region’s non-human colobus (Colobus guereza) (Figure 1). Chimpanzee samples were primates, including patas monkeys (Erythrocebus patas), mona collected from Kanyanchu, an area that has a habituated monkeys (Cercopithecus mona), and olive baboons (Papio anubis) chimpanzee community as a result of tourism (Figure 1). All [4,10,16]. However, previous research has indicated that O. samples were collected non-invasively immediately after defecation bifurcum is not commonly transmitted among primate species and placed into sterile tubes. Date, location, species, age and sex (including humans) because different parasite variants within the category, and social group membership were recorded. Human species are adapted to specific hosts [4,16,17]. samples were collected after the receipt of Institutional Review In Uganda, a number of primate species harbor Oesophagosto- Board-approved informed consent following World Health Orga- mum, as evidenced by microscopic detection of eggs in feces. These nisation protocols. Samples collection occurred in three villages: include members of the primate subfamilies Cercopithecinae and Ibura, Kanyansohera, and Kasojo, which are less than 5 km from Colobinae, as well as chimpanzees (Pan trogolodytes) [18–20]. There the border of the park (Figure 1). Individuals between the ages of 2 have also been reports of oesophagostomiasis in human patients in and 70 were suitable participants of this study. Consenting Uganda, although no such reports, to our knowledge, have been participants were given instructions on how to collect the sample, published since the 1980s [5,21], perhaps due to under-reporting which was then retrieved for processing within one day. or improvements in treatment. With the exception of chimpan- Samples were subjected to a modified ethyl acetate concentra- zees, which are infected with both O. bifurcum and O. stephanostomum tion method, recommended in the approved guidelines of the [22], the species of Oesophagostomum infecting Ugandan primates Clinical and Laboratory Standards Institute for the identification and humans remains unknown. of intestinal-tract parasites [32,33]. Concentration by sedimenta- In this study, we examined Oesophagostomum within the primate tion was performed in the field using one gram of undiluted feces community of Kibale National Park, Uganda, using a combination without fixture in formalin, as formalin is a known inhibitor of the of microscopic and molecular methods. Species-specific identifi- polymerase chain reaction [34]. All materials were sterilized prior cation of eggs by microscopy alone is difficult, because eggs are to use, and care was exercised throughout the procedure to similar morphologically to other STHs, including hookworms, prevent contamination. Sediments were left uncapped for two Trichostrongylus spp., and the ‘‘false hookworm’’ Ternidens deminutus hours after completion of the procedure to allow ethyl acetate that [5,9,23–25]. In other studies, coproculture of L3 larvae or may inhibit polymerase chain reaction (PCR) to volatilize. necropsy to isolate adult worms have been used to identify these Sediments were then suspended in 2 mL RNALater nucleotide parasites to species [25,26]. Here, we used molecular methods to stabilization solution (Sigma-Aldrich, St. Louis, MO, USA) and detect and sequence Oesophagostomum DNA directly from feces; frozen at 220uC until shipment to North America. such methods have proven informative for other similar studies [27,28]. In addition, we used phylogenetic comparative methods Microscopy to ascertain whether primate host traits explain variation in Thin smears from sedimented feces were used for microscopy prevalence of Oesophagostomum infection among host species. Our [35]. All eggs of the genus Oesophagostomum were identified at 106 sampling and analyses included nearby human populations to objective magnification on a Leica DM2500 light microscope. assess whether Oesophagostomum is a public health concern in the Data were recorded on size, shape, color and internal contents of region, as well the parasite’s local propensity for zoonotic eggs. Images were captured at 406 objective magnification of all transmission. specimens using an Infinity1 CMOS digital microscope camera

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eight species of Oesophagostomum were represented in the alignment. Other species of varying relatedness, including other members of the taxa Chabertiidae (Chabertia ovina, Accession No. JF680981; Ternidens deminutus, Accession No. HM067975), (Strongylus vulgaris [40]), and (Necator americanus [36], and Ancylostoma duodenale [41]) were also included. Priming regions were selected to be identical among all species of Oesophagostomum but divergent from the other genera. Primer ITS2-21 was highly specific as confirmed by sequencing, since all PCR products matched Oesophagostomum despite the fact that a number of other parasites (including Strongyloides, Necator and Trichuris), were identified in the same samples during microscopic examination. External PCR was performed in 25 mL volumes using the FailSafe System (Epicentre Biotenchnologies, Madison, WI, USA) with reactions containing 16FailSafe PCR PreMix with Buffer C, 1 Unit of FailSafe Enyme Mix, 2.5 picomoles of each primer (NC1 and NC2), and 1 mL of template. Reactions were cycled in a Bio- Rad CFX96 platform (Bio-Rad Laboratories, Hercules, CA, USA) with the following temperature profile: 94uC for 1 min; 45 cycles of 94uC for 15 sec, 50uC for 30 sec, 72uC for 90 sec; and a final extension at 72uC for 10 min. Internal PCR was performed in 25 mL volumes using the DyNAzyme DNA Polymerase Kit (Thermo Scientific, Asheville, NC, USA) with reactions containing 0.5 Units of DyNAzyme I DNA Polymerase, 16Buffer containing 1.5 mM MgCl2, 2.5 picomoles of each primer (OesophITS2-21 and NC2), and 1 mL of template. Reactions were cycled with the following temperature profile: 95uC for 1 min; 45 cycles of 95uC for 15 sec, 55uC for 30 sec, 70uC for 90 sec; and a final extension at 70uC for 5 min. Amplicons were electrophoresed on 1% agarose gels stained with ethidium bromide, and purified from gels using the Zymoclean Gel DNA Recovery Kit (Zymo Research Corporation, Figure 1. Map of sample collection sites in and around Kibale National Park, Uganda. Irvine, CA, USA) according to the manufacturer’s instructions. doi:10.1371/journal.pntd.0002641.g001 Products were Sanger sequenced in both directions using primers OesophITS2-21 and NC2 on ABI 3730xl DNA Analyzers (Applied Biosystems, Grand Island, NY, USA) at the University of and Infinity Camera v.6.2.0 software (Lumenera Corporation, Wisconsin-Madison Biotechnology Center DNA Sequencing Facil- Ottawa, ON, Canada). Samples were considered negative after the ity. Sequences were hand-edited and assembled using Sequencher entire sediment sample was scanned and no eggs were found. We v4.9 (Gene Codes Corporation, Ann Arbor, MI, USA) and all note that while identification of Oesophagostomum eggs was based on ambiguous bases were resolved by repeat PCR and re-sequencing, a rigorous set of characteristics, this genus cannot easily be as described above. All new sequences were deposited in GenBank, distinguished from hookworm infection by eggs alone. However, under Accession Numbers KF250585 - KF250660. hookworms have not been found in previous surveys of the gastrointestinal parasites of this primate community [19,20], Phylogenetic Analyses suggesting that eggs identified with morphological characteristics Sequences were aligned using the computer program ClustalX of both Oesophagostomum and hookworm were almost certainly [42] with minor manual adjustment. Published reference sequenc- Oesophagostomum. es were included to identify putative species (AF136575, Y11733, AF136576) and as outgroups (HQ844232, Y11738, Y11735, Molecular Methods Y10790, AJ006149), and were trimmed to the length of the newly DNA was extracted from 200 mL of sedimented feces using a generated sequences using Mesquite v.2.75 [43]. Trimmed ZR Fecal DNA MiniPrep Kit (Zymo Research Corporation, sequences yielded the same tree topology as did untrimmed Irvine, CA, USA), following manufacturer protocols. External sequences (by neighbor-joining method; results not shown), PCR was performed targeting the ribosomal internal transcribed suggesting that the amplified region was sufficient for taxonomic spacer 2 gene using primers NC1 (59-ACGTCTGGTT- discrimination. Phylogenetic trees were reconstructed using CAGGGTTGTT-39) and NC2 (59-TTAGTTTCTTTTCCTCC- maximum likelihood in MEGA v.5.05 [44] and the Hasegawa- GCT-39), which generated products that ranged in size from 280 Kishino-Yano substitution model [45]. Phylogenetic support was to 400 bp, suggesting that, as expected, the primer set detected a assessed using 1,000 bootstrap replicates. To estimate Oesophagos- number of parasitic helminths present in the samples [27,36]. tomum genetic diversity, percent nucleotide-level sequence identity Subsequently, an internal, semi-nested PCR generating amplicons among sequences was calculated as the uncorrected pairwise of predicted size 260 bp was performed using primer NC2 and proportion of nucleotide differences (p-distance) in MEGA v5.05 newly designed Oesophagostomum-specific primer, OesophITS2-21 [44]. (59-TGTRACACTGTTTGTCGAAC-39). Primer OesophITS2- 21 was generated by aligning publicly available sequences of the Statistical Analysis Oesophagostomum internal transcribed spacer 2 gene [26,36–39], and Diagnostic performance of microscopy versus PCR was estimat- GenBank accession numbers HQ283349, HQ844232]. In total, ed by calculating sensitivity (i.e., true positive rate) and specificity

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(i.e., true negative rate) using MedCalc v.12.5.0 (MedCalc prevalence by PCR in all three species (although sample sizes were Software, Ostend, Belgium). Prevalence of infection was calculated low in some cases; Table 1). as the number of samples found to be positive for Oesophagostomum PGLS indicated that terrestriality, maximum home range, divided by the total number of samples collected, with 95% maximum group size, percent time spent in polyspecific associa- confidence intervals calculated using the modified Wald method tions, and average female body mass were not significant [46]. To determine whether prevalence differed among primate univariate predictors of Oesophagostomum prevalence (all P.0.05 host species, a chi-square test was conducted in Quantitative from PGLS with lambda = ML; Table 2). However, log daily Parasitology v3.0 [47]. To explore variation in prevalence among travel explained nearly 55% of the variation in prevalence among hosts while controlling for their phylogenetic non-independence, a host species (P,0.05, R2 = 0.546, from PGLS with the ML phylogenetic least squared regression (PGLS) was conducted in R estimate of lambda = 0). In a multivariate model, both group size [48] using the ape [49] and caper [50] libraries. Prevalence of and log daily travel were significant predictors of prevalence, with Oesophagostomum was included as the dependent variable, and group size showing a negative relationship and log daily travel a various primate life history traits were independent variables: positive relationship (Table 2). This two-predictor model including terrestriality (predominantly terrestrial versus predominantly arbo- group size and daily travel explained over 75% of the variation in real), maximum home range [51–56], maximum group size Oesophagostomum prevalence among species (model P,0.01, [51,53,55,57–60], percentage time spent in polyspecific associa- R2 = 0.7701; Table 3). tions [61,62], average female body mass, and average daily travel From 222 positive samples, 76 were randomly selected for distance (the latter was log transformed since the relationship was sequencing to represent as even a number of positive samples per close to exponential) [55,56,62–64]. Humans were omitted from host species as possible. All 76 sequences most closely matched the PGLS analysis because many of these traits vary widely among published Oesophagostomum ITS-2 DNA sequences using the human populations, making accurate estimations problematic. BLASTn tool on the National Centre for Biotechnology Infor- To determine the degree to which each Oesophagostomum lineage mation website. Phylogenetic analysis resolved these sequences (i.e., taxonomic unit) identified by DNA sequencing was host into three clades (Figure 3). Clade 1 contained all 12 sequences restricted, we calculated the phylogenetic dispersion of infected from olive baboons, one sequence from l’hoest monkeys, one hosts using the net relatedness index (NRI) in R [48] using the ape sequence from grey-cheeked mangabeys, three sequences from red [49] and picante libraries. Mean pairwise distance (MPD) was colobus and one sequence from red-tailed guenons. These weighted by the ratio of occurrence of each Oesophagostomum within sequences were identical to published reference sequences for O. each lineage, and compared to null expectation in 1000 randomly bifurcum [26,36]. Five additional sequences from l’hoest monkeys assembled communities. Results are reported as standard effects sorted into clade 1 and were 97.1% similar to this same O. bifurcum sizes, with values close to 1 indicating phylogenetic evenness (i.e., reference sequence. Clade 2 contained all eight sequences from Oesophagostomum lineages infect a greater diversity of hosts than chimpanzees, five sequences from blue monkeys, two sequences would be expected by chance), while values ,0.05 indicate from black and white colobus, two sequences from grey-cheeked phlylogenetic clustering (i.e., Oesophagostomum lineages are host- mangabeys, three sequences from red colobus, and twelve specific). sequences from red-tailed guenons. All sequences in clade 2 were identical to an O. stephanostomum reference sequence [26]. Clade 3 Results was composed of two nearly identical branches (99.4% identity) that contained all six sequences from humans, as well as sequences A total of 318 fecal samples from primates, including humans, from three blue monkeys, three black and white colobus, five grey- were collected (Table 1). Of these, 112 were positive for cheeked mangabeys, two red colobus, and two red-tailed guenons. Oesophagostomum by microscopy, for a community-wide prevalence These sequences were 92.4–93.0% and 93.0–93.6% similar to O. of infection of 35.2% (Table 1). All eggs identified by microscopy bifurcum, and O. stephanostomum, respectively, but were not identical were similar in internal and external morphology in samples from to any published reference sequence. all primate species (Figure 2). Eggs were 65–80 by 35–50 mmin Host species were phylogenetically clustered within O. bifurcum size, which is consistent with previous results from this community clade 1 (NRI = 21.76, P,0.05). Clade 2 (O. stephanostomum) did not [19,20] (Figure 2). vary significantly from the null expectation of no host clustering, PCR generated single, clear amplicons of expected size (260 bp) NRI = 0.86, P = 0.75). Clade 3 was marginally phylogenetically in 222 samples, indicating positive detection of Oesophagostomum over-dispersed with respect to distribution of host species DNA, for an overall prevalence of 69.8%. No amplicons were (NRI = 1.24, P = 0.04). present in remaining samples. Resulting DNA sequences over- lapped 100% with published sequences and contained no Discussion insertions or deletions, making alignment unequivocal. When PCR results were compared to microscopy, the overall Here we evaluate the prevalence of Oesophagostomum infection in sensitivity of PCR was 100% (95% CI 96.8%–100.0%), but wild primates and humans in Western Uganda using both specificity was only 47.5% (95% CI 40.5%–54.7%). Thus, PCR microscopy and PCR. Our results clearly show that prevalence did not classify any microscopy-positive samples as negative but varied significantly among host species. Humans had the lowest identified 109 microscopy-negative samples as positive. prevalence of infection likely because of avoidance behaviors such Prevalence of Oesophagostomum infection (as determined by both as sanitation practices [65,66] and because of the common use of microscopy and PCR) varied significantly among host species antihelminthics in the region. Red colobus and black and white (microscopy: chi-square = 54.31, df = 8, P,0.0001; PCR: chi- colobus also had comparatively low prevalence of infection, as square = 112.2, df = 8, P,0.0001). Both microscopy and PCR found in previous studies [16,20,67]. This observation may reflect identified humans as having the lowest prevalence of infection colobine gastrointestinal physiology, which is characterized by (8.3% and 25.0%, respectively), followed by red colobus (17.2% folivory and foregut fermentation [68], and the associated regular and 40.6%, respectively). Chimpanzees, l’hoest monkeys, and olive ingestion of plant secondary compounds that may suppress baboons had the highest prevalence by both methods, with 100% infection by pathogenic organisms [69]. Conversely, the high

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Figure 2. Microscopic images of representative Oesophagostomum sp. eggs found in the feces of infected primate hosts. Images were captured at 406 objective magnification from thin smears of sedimented feces. A = blue monkey, B = black and white colobus, C = chimpanzee, D = l’hoest monkey, E = grey-cheeked mangabey, F = olive , G = red-tailed guenon, H = human. All eggs were between 65 and 84 mm long and between 35 and 55 mm wide. doi:10.1371/journal.pntd.0002641.g002 prevalence of infection in chimpanzees, olive baboons, and l’hoest the opposite in the case of Oesophagostomum: smaller primate groups monkeys may reflect reduced physiological barriers to infection or with large daily travel distances had higher prevalence. increased susceptibility. To explain this interspecific variation in with larger day ranges may encounter greater habitat variation [73], prevalence, we examined correlations between life history which may increase exposure to Oesophagostomum from environmen- variables and prevalence among host species. We found that two tal sources. In addition, previous research has implicated terrestri- variables, daily travel distance and group size, explain over 75% of ality as an important factor affecting the prevalence of trematode the variance in Oesophagostomum prevalence among host species. parasites in primates [74]. In our study, the three host species with Surprisingly, body mass, the strongest predictor of helminth highest Oesophagostomum prevalence (chimpanzees, olive baboons species richness elsewhere, was not significant here [67]. and l’hoest monkeys) were also the only three predominantly Previous studies have concluded that group-living animals with terrestrial species. Although this trend was not statistically small home ranges are likely to suffer high intensities of infection due significant, it is possible that terrestrial primates contact soil more to frequent environmental re-exposure [70–72]. Our results indicate frequently, and thus the infective stages of STHs.

Table 1. Prevalence of Oesophagostomum spp. in nine primate host species (including humans) in and near Kibale National Park, Uganda, based on microscopy and PCR.

Number positive Prevalence (95% CI)

Species N Microscopy PCR Microscopy PCR

BM (Blue monkey) 33 10 24 30.3 (17–47) 72.7 (56–85) BW (Black and white colobus) 37 8 21 21.6 (11–37) 56.8 (41–71) CH (Chimpanzee) 30 18 30 60.0 (42–75) 100 (86–100) GM (Gray-cheeked mangabey) 42 17 39 40.5 (27–56) 92.9 (80–98) HU (Human) 36 3 9 8.3 (2–23) 25.0 (14–41) LM (L’hoest monkey) 8 6 8 75.0 (40–94) 100 (63–100) OB (Olive baboon) 27 18 27 66.7 (48–81) 100 (85–100) RC (Red colobus) 64 11 26 17.2 (10–28) 40.6 (29–53) RT (Red-tailed guenon) 41 21 38 51.2 (36–66) 92.7 (80–98) TOTAL 318 112 222 35.2 (30–40) 69.8 (65–75)

doi:10.1371/journal.pntd.0002641.t001

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Table 2. Phylogenetic generalized least-squared multiple analysis, we identified both O. bifurcum and O. stephanostomum in the regression models of the relationship between Kibale primate community. However, we found only O. Oesophagostomum prevalence and univariate life-history stephanostomum in chimpanzees, although the possibility of unde- variables of diurnal primates (excluding humans) within the tected O. bifurcum infections cannot be ruled out. Kibale community. In addition, we identified a third Oesophagostomum lineage that did not cluster with any published sequence and thus may represent a previously uncharacterized taxon. It is possible that Univariate Model l Slope F P Adjusted r2 this new taxon has remained undetected in previous molecular investigations. We examined the OB primer that has been used Terrestriality 0.97 0.20495 2.93 0.130 0.2159 previously to identify O. bifurcum [36] and conclude that it would Home range 1.00 0.00004 1.07 0.400 0.0101 probably not amplify our newly identified taxon due to Group size 1.00 20.00023 0.05 0.950 20.1567 mismatched bases at both the 59 and 39 ends of the primer. It is Polyspecific association 1.00 20.19713 1.16 0.375 0.0225 therefore possible that the new taxon we identified exists elsewhere Body mass 1.00 0.00400 0.61 0.578 20.0590 (e.g. in Togo and Ghana) but has been not been detected or differentiated from other members of the genus. However, we Log daily travel 0.00 0.18500 9.43 0.014 0.5464 caution that these inferences are based on a short region of a single doi:10.1371/journal.pntd.0002641.t002 gene, and that sequencing additional genes as well as morpholog- ical characterization of L3 larvae and adults will be necessary to Although group size was not a significant predictor of confirm these findings. Nonetheless, our results suggest a prevalence in univariate analyses, our multivariate analysis found heretofore unappreciated degree of hidden genetic diversity within smaller groups with large daily travel distances to be at greatest this well-described genus of parasites that are known to infect risk of infection. This finding contrasts with previous studies humans. showing that increased intragroup contact increases exposure Interestingly, all Oesophagostomum sequences recovered from [71,75]. In Kibale, positive associations between group size and humans clustered with the previously undescribed third taxon, parasite richness have been documented for protozoan parasites in and not with published O. bifurcum sequence from humans mangabeys [76]. However parasite richness is not necessarily elsewhere in Africa (clade 1) [36]. In Ghana, geographic associated with prevalence. Small primate groups might maintain separation between humans and non-human primates infected Oesophagostomum high intra-group infection rates for certain parasites if transmission with , despite apparently conducive environments for zoonotic transmission, motivated efforts to determine the host within the group is frequent, thus maintaining high prevalence (as range of the parasite using molecular methods [28]. Genome-wide seen here) without correspondingly high parasite richness. analyses (amplified fragment length polymorphism, random Our study detected substantial cryptic phylogenetic diversity in amplification of polymorphic DNA) suggested that O. bifurcum Oesophagostomum infecting Ugandan primates. Currently, the clusters into distinct groups by host species, thus suggesting that principal human Oesophagostomum species is considered to be O. zoonotic transmission is uncommon [4,17]. By contrast, in our bifurcum [5], while other great apes harbor O. stephanostomum study area, no such geographic separation exists between humans [6,12,18,77]. Recently, however, chimpanzees inhabiting a and non-human primates. In this setting, we found that both northern sector of Kibale were identified as positive for O. humans and non-human primates were infected with the novel bifurcum, making this the first discovery of O. bifurcum in non- Oesophagostomum clade 3, which is phylogenetically over-dispersed human apes. The same study identified chimpanzees also infected compared to the other Oesophagostomum clades. While our or co-infected with O. stephanostomum [22]. In our phylogenetic conclusions await verification from more detailed examination of the Oesophagostomum genome, our results nevertheless suggest that this novel clade may be broadly transmissible among species of Table 3. Phylogenetic generalized least-squared multiple distantly related primate hosts, including humans. The Kibale regression models of the relationship between ecosystem is known for its high degree of spatio-temporal overlap Oesophagostomum prevalence and life-history variables of between humans and non-human primates and its ensuing high diurnal primates (excluding humans) within the Kibale rates of transmission of diverse pathogens across primate species community. [78–81]. Our results provide further evidence for cross-species pathogen transmission between wild primates and humans in this region. Multivariate P Adjusted Model l Slope t P trait F overall r2 Our paired analyses applying both microscopy and PCR to the same samples indicate that traditional methods based on y-intercept 0.00 21.93390 22.65 0.045 9.95 0.015 0.7189 microscopy may significantly underestimate prevalence. Concen- Home range + 0.00012 22.16 0.083 tration methods followed by microscopic visualisation of eggs in Log daily travel 0.00039 3.70 0.014 thin smears are considered definitive diagnostic methods for soil- y-intercept 0.00 20.89378 22.58 0.049 12.730.009 0.7701 transmitted helminth (STH) infections [82,83]. Previous studies Group size + 20.00180 22.61 0.047 that have used fecal sedimentation and microscopy have reported Oesophagostomum infection prevalence estimates between approxi- Log daily travel 0.24894 5.04 0.004 mately 3% and 10% in wild Ugandan primates [19,20]. These y-intercept 0.00 21.48140 21.97 0.120 8.39 0.032 0.7599 values are considerably lower than what we report here using Home range + 20.00006 20.88 0.425 molecular methods; however, our results parallel other studies that Group size + 20.00127 21.36 0.245 have estimated prevalence using molecular methods [16,22]. Not Log daily travel 0.33085 3.14 0.035 surprisingly, we find that PCR is more sensitive than microscopy, perhaps because it can detect Oesophagostomum infection even when doi:10.1371/journal.pntd.0002641.t003 eggs are not present. For example, tissues or secretions shed by

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Figure 3. Phylogenetic analysis of Oesophagostomum based on ITS2 rDNA (260 bp) sequences. Nucleotide sequences were aligned using Clustal X software [42]. Phylogenetic relationships were inferred in MEGA5 [44], using the maximum likelihood method with a Hasegawa-Kishino- Yano model of nucleotide substitution [45]. The best-scoring maximum-likelihood tree is shown here (2lnL = 656.5). Bootstrap values (%) greater than 50% are shown. Taxon names of sequences generated in this study are in bold and correspond to the host species followed by the number of infected individuals in parentheses (BM = blue monkey, BW = black and white colobus, CH = chimpanzee, LM = l’hoest monkey, GM = grey-cheeked mangabey, OB = olive baboon, RC = red colobus, RT = red-tailed guenon, and HU = human). Reference sequences correspond to Genbank accession numbers AF136575 and Y11733 for O. bifurcum, AF136576 for O. stephanostomum, HQ844232 for O. sp, Y11738 for O. quadrispinulatum, Y11735 for O. dentatum, Y10790 for O. venulosum, and AJ006149 for O. radiatum. Scale bar indicates nucleotide substitutions per site. doi:10.1371/journal.pntd.0002641.g003 adult worms into the intestinal lumen would be detected by PCR, pathogen of concern beyond its accepted foci of infection in Togo as would eggs that have hatched into L1 larva prior to fixation and Ghana, and perhaps across all of equatorial Africa. during the sedimentation procedure. To our knowledge, ours is the first study in several decades to Acknowledgments report human Oesophagostomum infection in Uganda, a country that is over 3,000 km from known foci of infection in West Africa [5]. We gratefully acknowledge the Uganda Wildlife Authority and the Uganda Given that the prevalence of Oesophagostomum was 25% in our National Council for Science and Technology for granting permission to conduct this research. We also thank Dennis Twinomugisha and Lauren sample of people, we suspect that this parasitic infection occurs Chapman for assistance with logistics, Chesley Walsh, Robert Basaija, more commonly across Sub-Saharan Africa than previously Peter Tuhairwe, and Richard Kaseregenyi for assistance in the field, thought and may be causing infections that are untreated or Dwight Bowman for assistance with parasitological identifications, Aleia misdiagnosed. Our finding of a previously genetically unchar- McCord and Mary Thurber for laboratory training, Johanna Bleecker for acterized lineage of Oesophagostomum that may be transmitted map preparation, and Tavis Anderson, Maxwell Farrell, and Chelsey among primate species underscores that the diversity (genetic and Chisholm for analytical advice. otherwise) of this parasite genus may be under-sampled in Africa. Further ecological studies of Oesophagostomum in Uganda and Author Contributions elsewhere are needed to quantify the degree of enzootic versus Conceived and designed the experiments: RRG CAC TJD TLG. zoonotic transmission. Regardless of the outcome of such research, Performed the experiments: RRG CAC PAO TLG. Analyzed the data: our results suggest that Oesophagsotomum should be considered a RRG TJD TLG. Wrote the paper: RRG TJD TLG.

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